Neurons are important cells in the nervous system, being involved in receiving, organizing, and transmitting information. Each neuron contains a cell body, an axon (a thin, tube-like process that arises from the cell body and travels some distance before terminating), and dendrites (neuronal processes of the cell body that are shorter and thicker than axons). The cytoskeleton of the neuron provides mechanical strength to the axons and dendrites and a track for transport of materials between the cell body and the nerve terminal. The cytoskeleton is a system of interconnected macromolecular filaments. Three polymeric structures form the basis of this cytoskeleton: actin filaments (microfilaments), microtubules, and intermediate filaments.
Intermediate filaments (IFs) are 10 nm filaments that are found in most eukaryotic cells. There are six classes of IFs recognized according to sequence homology and gene structure: type I and II IFs include the acidic, neutral, and basic keratins; type III IFs include vimentin, desmin, the glial fibrillary acidic protein (GFAP), peripherin, and plasticin; type IV IFs include neurofilament proteins and .alpha.-internexin; type V IFs include the nuclear lamins; and type VI IFs include nestin expressed in neuroepithelial cells.
Neuronal intermediate filaments (NIFs) include peripherin, .alpha.-internexin and neurofilaments. The NIF proteins are encoded by a large multigene family displaying cell and tissue-specific expression patterns throughout development. For example, during embryonic development in neurons, .alpha.-internexin and peripherin are expressed at high levels, which decline as the levels of neurofilament proteins increase in the postnatal period during maturation of nerve cells.
The NIF proteins are made up of an assembly of protein subunits. The current model of NIF assembly involves 1) the bonding of two subunits to form a dimer; 2) the aggregation of two antiparallel dimers to form a tetramer, called a protofilament; 3) the joining of about eight protofilaments end on end; and 4) the association of these joined protofilaments to other joined protofilaments by staggered overlaps to form a 10 nm filament. Moreover, the cytoplasmic NIF proteins share a homologous central region of similar size (approximately 310 amino acids) flanked by amino- and carboxy-terminal domains varying greatly in sequence and in length. The central region of NIF proteins forms an extended .alpha.-helical rod domain that plays a critical role in protein assembly into 10 nm filaments.
Of all the NIF proteins that participate in the formation of the neuronal cytoskeleton, the neurofilament triplet proteins are the most abundant. These neurofilament proteins (NFs) are expressed exclusively in neurons. NFs are found predominantly in axons, where they run longitudinally and parallel to each other. NFs are formed by the copolymerization of three NF protein subunits: light (61 kDa)(NF-L), medium (90 kDa)(NF-M), and heavy (110 kDa)(NF-H). NF-L subunits form the core of the NF and are essential for NF assembly. NF-M and NF-H subunits form side-arm projections in the NF structure, cross-linking NFs and other neuronal structures into a three-dimensional IF matrix. The NF-M and NF-H projections appear to modulate the spacing between NFs, regulating the caliber of axons.
The three different NF subunits are encoded by three different genes, each of which is under separate developmental control. Significant expression of NF-L and NF-M is first seen in the embryonic brain, while in most neurons, NF-H expression is delayed relative to the other subunits and occurs in the post-natal period.
Neurofilaments and Neurodegenerative Diseases:
Neurofilaments have been linked to a number of neurodegenerative diseases. Large motor neurons are particularly vulnerable to NF abnormalities because of their high NF content and their long axons. Abnormal depositions of NFs is a phenomenon observed in many neurodegenerative diseases (Table 1).
TABLE 1 ______________________________________ Human Diseases with Abnormal NF Accumulations Disease Abnormalities Prevalence ______________________________________ ALS NF depositions in motor neurons 70% of cases Decline of 60% in NF-L mRNA Parkinson's disease Lewy bodies in substantia nigra 100% of cases and locus coreuleus Declines of 30% NF-L mRNA and 70% NF-H mRNA Alzheimer's disease Cortical Lewy bodies 20% of cases Decline of 70% in NF-L mRNA Lewy body Dementia Cortical Lewy bodies Guam-Parkinsonism NF depositions in motor neurons 100% of cases Giant Axonal NF accumulations in peripheral Neuropathy axons Peripheral NF accumulations in peripheral Neuropathies axons that can be induced by various toxic agents, such as IDPN, hexanedione, acrylamide ______________________________________
As an example, there is evidence that deregulation of neurofilament expression may play a central role in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). ALS is an adult-onset neurological disorder resulting from the degeneration of motor neurons in the brain and spinal cord. This leads to denervation atrophy of skeletal muscles and, ultimately, to paralysis and death.
A characteristic pathological finding in ALS patients is the presence of abnormal neurofilament accumulation in the axons of spinal motor neurons. Evidence suggests that NF accumulation may cause axonal degeneration by impeding transport of components required for axonal maintenance. Aberrant neuronal swellings that are highly reminiscent of those found in ALS have recently been reported in transgenic mice overexpressing either the NF-L or NF-H gene.
As additional evidence for NF involvement in ALS, a recent report has revealed that there is a 60% decrease in levels of NF-L mRNA in the motor neurons of patients with ALS (Bergeron et al., (1994) Brain Res. 659:272-276). As well, mutant NF-H alleles have been found in some ALS patients (Figlewicz et al. (1994) Hum. Molec. Genet. 3:1757-1761).
NFs are also implicated in Parkinson's disease. The pathological hallmark of idiopathic Parkinson's disease is the presence of Lewy bodies (LBs), cytoplasmic inclusions made up of altered NF proteins. These LBs are located in neurons of the substantia nigra. A subset of demented elderly patients also exhibit LB-like inclusions in their cortical neurons. The mechanisms involved in the abnormal aggregation of NF proteins to form LBs are still unknown. It has been found that levels of NF-L and NF-H mRNAs in substantia nigra neurons are reduced in Parkinsonian patients as compared to age-matched controls. There is also reduced NF synthesis in LB-containing neurons.
In patients with Alzheimer'disease, cortical LBs are present in approximately 20% of cases. It has also been discovered that there is a 70% decrease in NF-L mRNA expression in these patients. (Crapper McLachlan et al., (1988) Molec. Brain Res. 3:255-262)
The mechanisms underlying the abnormal aggregation of NF proteins in disease are still unknown. It is very interesting, however, that decreased levels of NF mRNAs are associated with degenerative neurons in ALS, Parkinson's disease, Alzheimer's disease, and other neurodegenerative diseases. Whether down-regulating NF gene expression can directly contribute to formation of NF depositions or to other pathological changes in specific populations of neurons remains to be determined.
Neurofilaments and Aging:
Aging is a factor that may contribute to axonal atrophy. There is a normal decline (50-60%) in NF mRNA expression with aging. (Parhad et al., (1995) J. Neurosci. Res. 41:355-366) The resulting decrease in NFs may be linked to axonal atrophy and a reduced capacity for compensatory axonal outgrowth during aging. Methods of enhancing neuronal regeneration could attenuate the aging process.
Neurofilaments and Injury:
Following injury in mammals, peripheral nervous system (PNS) axons have the capacity to regenerate, whereas central nervous system (CNS) neurons have limited axonal outgrowth. It is widely believed that NFs are not required for axonal regeneration following injury. This notion is based on the observation that neurofilament mRNAs decrease two to threefold following axotomy. Although NFs are present in the CNS, their numbers, which are much lower than in the PNS, may not be sufficient to sustain axonal outgrowth.
Animal Models:
A number of animal models for neurodegenerative diseases have been created. Transgenic mice have been generated that overexpress NF genes. In one instance, transgenic mice carrying multiple copies of the human NF-H gene were created (F. Cote, J. -F. Collard, and J. -P. Julien, (1993) Cell 73:35--46; J. -F. Collard, F. Cote, and J. -P. Julien, (1995) Nature 375:61-64). These mice progressively develop physiological and pathological features reminiscent of ALS. They are characterized by the presence of abnormal NF accumulations in the perikarya and proximal axons of spinal motor neurons. With age, motor dysfunction progresses by the atrophy and subsequent degeneration of axons distal to the NF swellings. As well, there is a dramatic reduction in rates of axonal transport of all NF proteins and other proteins, including tubulin and actin.
Other transgenic mice have been created that overproduce normal and mutant NF-L proteins (Xu et al. (1993) Cell 73:23-33; Lee et al. (1994) Neuron 13:975:988). These mice develop motor neuron degeneration accompanied by the accumulation of neurofilaments in spinal motor neurons.
A transgenic mouse has been created that expresses a fusion protein in which the carboxyl terminus of NF-H was replaced by beta-galactodisase (J. Eyer and A. Peterson, (1994) Neuron 12:389-405). The resulting axons develop small calibers.
A transgenic mouse has been engineered that expresses a mutant form of the human copper-zinc superoxide dismutase (SOD)(Gurney et al., (1994) Science 264:1772-1775). This mutation is responsible for 2% of ALS cases. The mechanism by which SOD mutants induce selective vulnerability to degeneration of motor neurons in familial ALS is unknown; however, NF accumulation and abnormalities in degenerating motor neurons have been reported.
Transgenic mouse models that overexpress wild-type or mutant NF genes have provided evidence that NF accumulations can play a causal role in motor neuron disease. It seems paradoxical, however, that levels of NF mRNAs are not increased; rather, they are reduced in patients with neurodegenerative diseases, including ALS, Parkinson's disease, and Alzheimer's disease. This suggests that NF depositions in neurodegenerative diseases might be induced by perturbations in the stoichiometry of assembled NF subunits; accordingly, there is a need for an animal model having disrupted NF genes.
A number of animal models having decreased amounts of neurofilaments have been reported. A mutant strain of quail has been discovered that is lacking NF-L subunits and is deficient in NFs (Yamasaki et al (1992) Lab. Invest. 66:734-743; Yamasaki et al. (1991) Acta Neuropathol. 82:427-434). As well, a canine model of motor neuron disease has been shown to have NF accumulations in motor neurons and a low ratio of NF-L to NF-H mRNAs (Murna and Cork, (1993) Lab Invest. 69:436-442).
Homologous recombination may be employed for inactivation or alteration of genes in a site-directed manner. A number of papers describe the use of homologous recombination in mammalian cells, including human cells. Illustrative of these papers are Kucherlpati et al. (1984) Proc. Natl. Acad. Sci. USA 81:3153-3157; Kucherlapati et al. (1985) Mol. Cell. Bio. 5:714-720; Smithies et al. (1985) Nature 317:230-234; Wake et al. (1985) Mol. Cell. Bio. 8:2080-2089; Ayares et al. (1985) Genetics 111:375-388; Ayares et al. (1986) Mol. Cell. Bio. 7:1656-1662; Song et al. (1987) Proc. Natl. Acad. Sci. USA 84:6820-6824; Thomas et al. (1986) Cell 44:419-428; Thomas and Capecchi (1987) Cell 51:503-512; Nandi et al. (1988) Proc. Natl. Acad. Sci. USA 85:3845-3849; and Mansour et al. (1988) Nature 336:348-352. Various aspects of using homologous recombination to create specific genetic mutations in embryonic stem cells and to transfer these mutations to the germline have been described (Evans and Kaufman (1981) Nature 294:154-146; Doctschman et al., (1987) Nature 330:576-578; Thomas and Capecchi (1987) Cell 51:503-512; Thompson et al. (1989) Cell 56:316-321. The combination of a mutant polyoma enhancer and a thymidine kinase promoter to drive the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney and Donerly, Cell 35:693-699, 1983.
There are, however, no animal models in which the different NF subunits are completely absent. The present invention is directed towards overcoming this void. The role of each NF subunit in the assembly and function of NFs is poorly understood. A significant need remains for an animal model to determine the specific role of each NF subunit in neuronal structure and function, to test the possibility that perturbations in the stoichiometry of NF subunits might induce abnormal NF depositions, and to further investigate the mechanisms underlying NF-induced pathology.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incorporated by reference in their entireties in this application.